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Early p53 mutations in nondysplastic Barrett's tissue detected by the restriction site mutation (RSM) methodology.

Jenkins GJ, Doak SH, Griffiths AP, Tofazzal N, Shah V, Baxter JN, Parry JM - Br. J. Cancer (2003)

Bottom Line: Tissue samples taken from these patients (93 samples in total) were analysed for the presence of low-frequency p53 mutations at hotspot codons: 175, 213, 248, 249, 282.Although no statistically significant associations were found, p53 mutations reflected histological progression in Barrett's patients with p53 mutations found in 30% of metaplasia patients (P=0.4) and low-grade dysplasia patients (P=0.33) and 45% of high-grade dysplasia patients (P=0.15).Detected p53 mutations were mainly GC to AT transitions at CpG sites.

View Article: PubMed Central - PubMed

Affiliation: Swansea Clinical School, University of Wales Swansea, UK. g.j.jenkins@swansea.ac.uk

ABSTRACT
Barrett's oesophagus is a premalignant condition whose incidence is rising dramatically. Molecular markers are urgently needed to identify Barrett's patients at the highest risk of cancer progression. To this end, we have used a rapid molecular technique, restriction site mutation (RSM), to detect low-frequency mutations in the p53 tumour suppressor gene in premalignant Barrett's tissues of cancer-free patients. In total, 38 endoscopically diagnosed Barrett's patients with a range of histological stages of Barrett's progression, plus four control patients without Barrett's oesophagus, were analysed for early p53 mutations. Tissue samples taken from these patients (93 samples in total) were analysed for the presence of low-frequency p53 mutations at hotspot codons: 175, 213, 248, 249, 282. In total, 13 of the 38 Barrett's patients were shown to possess a p53 mutation in at least one sample (no mutations in the four control patients). Although no statistically significant associations were found, p53 mutations reflected histological progression in Barrett's patients with p53 mutations found in 30% of metaplasia patients (P=0.4) and low-grade dysplasia patients (P=0.33) and 45% of high-grade dysplasia patients (P=0.15). Detected p53 mutations were mainly GC to AT transitions at CpG sites.

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Related in: MedlinePlus

Example of an RSM experiment involving the Msp I restriction enzyme of codon 248 and DNA extracted from nine Barrett's tissues. The right-hand lane contains a positive control for the PCR step and shows the expected PCR band size. The left-hand lane contains a DNA ladder showing the 100, 200 and 300 bp bands. The central lanes contain DNA from Barrett's biopsies subject to RSM analysis. The highlighted lane contains an undigested band of the correct size and was subject to DNA sequencing where it was confirmed that it contained a GC to AT mutation. The remaining lanes (including the internal digestion control situated next to the PCR-positive control) have digested and hence do not contain mutations in this restriction site (codon 248).
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fig1: Example of an RSM experiment involving the Msp I restriction enzyme of codon 248 and DNA extracted from nine Barrett's tissues. The right-hand lane contains a positive control for the PCR step and shows the expected PCR band size. The left-hand lane contains a DNA ladder showing the 100, 200 and 300 bp bands. The central lanes contain DNA from Barrett's biopsies subject to RSM analysis. The highlighted lane contains an undigested band of the correct size and was subject to DNA sequencing where it was confirmed that it contained a GC to AT mutation. The remaining lanes (including the internal digestion control situated next to the PCR-positive control) have digested and hence do not contain mutations in this restriction site (codon 248).

Mentions: The RSM products were then visualised on 5% polyacrylamide gels (Biorad, Hemel Hempstead, UK) and poststained with silver. Restriction enzyme-resistant RSM products were detected on the gels as undigested bands of the correct size, by comparison to the PCR-positive control (see Figure 1Figure 1


Early p53 mutations in nondysplastic Barrett's tissue detected by the restriction site mutation (RSM) methodology.

Jenkins GJ, Doak SH, Griffiths AP, Tofazzal N, Shah V, Baxter JN, Parry JM - Br. J. Cancer (2003)

Example of an RSM experiment involving the Msp I restriction enzyme of codon 248 and DNA extracted from nine Barrett's tissues. The right-hand lane contains a positive control for the PCR step and shows the expected PCR band size. The left-hand lane contains a DNA ladder showing the 100, 200 and 300 bp bands. The central lanes contain DNA from Barrett's biopsies subject to RSM analysis. The highlighted lane contains an undigested band of the correct size and was subject to DNA sequencing where it was confirmed that it contained a GC to AT mutation. The remaining lanes (including the internal digestion control situated next to the PCR-positive control) have digested and hence do not contain mutations in this restriction site (codon 248).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2747568&req=5

fig1: Example of an RSM experiment involving the Msp I restriction enzyme of codon 248 and DNA extracted from nine Barrett's tissues. The right-hand lane contains a positive control for the PCR step and shows the expected PCR band size. The left-hand lane contains a DNA ladder showing the 100, 200 and 300 bp bands. The central lanes contain DNA from Barrett's biopsies subject to RSM analysis. The highlighted lane contains an undigested band of the correct size and was subject to DNA sequencing where it was confirmed that it contained a GC to AT mutation. The remaining lanes (including the internal digestion control situated next to the PCR-positive control) have digested and hence do not contain mutations in this restriction site (codon 248).
Mentions: The RSM products were then visualised on 5% polyacrylamide gels (Biorad, Hemel Hempstead, UK) and poststained with silver. Restriction enzyme-resistant RSM products were detected on the gels as undigested bands of the correct size, by comparison to the PCR-positive control (see Figure 1Figure 1

Bottom Line: Tissue samples taken from these patients (93 samples in total) were analysed for the presence of low-frequency p53 mutations at hotspot codons: 175, 213, 248, 249, 282.Although no statistically significant associations were found, p53 mutations reflected histological progression in Barrett's patients with p53 mutations found in 30% of metaplasia patients (P=0.4) and low-grade dysplasia patients (P=0.33) and 45% of high-grade dysplasia patients (P=0.15).Detected p53 mutations were mainly GC to AT transitions at CpG sites.

View Article: PubMed Central - PubMed

Affiliation: Swansea Clinical School, University of Wales Swansea, UK. g.j.jenkins@swansea.ac.uk

ABSTRACT
Barrett's oesophagus is a premalignant condition whose incidence is rising dramatically. Molecular markers are urgently needed to identify Barrett's patients at the highest risk of cancer progression. To this end, we have used a rapid molecular technique, restriction site mutation (RSM), to detect low-frequency mutations in the p53 tumour suppressor gene in premalignant Barrett's tissues of cancer-free patients. In total, 38 endoscopically diagnosed Barrett's patients with a range of histological stages of Barrett's progression, plus four control patients without Barrett's oesophagus, were analysed for early p53 mutations. Tissue samples taken from these patients (93 samples in total) were analysed for the presence of low-frequency p53 mutations at hotspot codons: 175, 213, 248, 249, 282. In total, 13 of the 38 Barrett's patients were shown to possess a p53 mutation in at least one sample (no mutations in the four control patients). Although no statistically significant associations were found, p53 mutations reflected histological progression in Barrett's patients with p53 mutations found in 30% of metaplasia patients (P=0.4) and low-grade dysplasia patients (P=0.33) and 45% of high-grade dysplasia patients (P=0.15). Detected p53 mutations were mainly GC to AT transitions at CpG sites.

Show MeSH
Related in: MedlinePlus